The long-term objective of these studies is to elucidate the mechanisms that mediate cleavage of intranuclear concatameric herpes simplex virus (HSV) DNA, and insertion of cleaved genomes into preformed capsids. This process is the target of several novel antiviral drugs and understanding the process may lead to modified drugs with broader specificities. The packaging reaction can be viewed as an interaction of two components of a molecular motor. One part receives the DNA (the procapsid), and the second part, the terminase, processes concatameric DNA and drives it into the capsid. Studies focus on members of a protein complex comprised of pULl5, pUL28 and pUL33 that we have identified in infected cells and that have been shown to be required for DNA packaging and are capsid components. The first specific aim is to identify and characterize the ATPase responsible for DNA cleavage and packaging. UL15 is the strongest candidate for fulfilling such a role, although proteins that interact with UL15 may also be involved. Purified proteins will be tested for ATPase activity and mutants lacking the activity will be tested for the ability to support viral replication. The second specific aim seeks to identify and characterize DNA binding relevant to DNA packaging. The UL28 protein has been shown in vitro to bind Pac1 DNA (a sequence that is necessary for formation of the terminus of the short component of viral DNA). Efforts will be made to determine if this occurs in vivo. Efforts will also seek to identify proteins that associate with Pac2, a sequence required to initiate the packaging reaction at the terminus of the long component of viral DNA. To determine if the in vitro activities are relevant in vivo, mutants lacking DNA binding activities will be tested for their ability to support viral DNA cleavage/packaging and replication. The third specific aim will seek to determine the morphology of the molecular motor that drives DNA packaging and thus, will provide considerable information about how the machine functions. Evidence supports the possibility that the UL6 protein (pUL6) forms the pore into which DNA is inserted and that pUL15 may bind this pore. Efforts are described to determine the localization in capsids of components of the UL15, UL28, and UL33. At least pUL15 is hypothesized to interact with pUL6 to mediate docking of the terminase with the procapsid, so should be present on the external surface of the capsid as revealed by electron microscopic immunogold analysis. [unreadable] [unreadable] [unreadable]